User:DavidC/16 September 2009
From 2009.igem.org
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Wednesday the 16th
DNA extraction
DNA extraction from bio-brick R0040+B0030 and P1002+C0040 from 500mL of E.coli DH5alpha growth in LB medium.
Ligation: samples digested on the 15th of September
Ligation pairs: plasmid / insert
BBa_B0014 / BBa_I712669 ;
BBa_P1003/BBa_B0014 ;
BBa_J23100/BBa_B0014
First report:
Plasmid = 1µL
Insert = 6µL
Second report:
Plasmid = 3µL
Insert = 5µL
Third report:
Plasmid = 1µL
Insert = 5µL
NEB Enzymes
For each samples add sterilized water to obtain a maximum volume equals to 8µL.
Ligation mix (NEB):
9µL of T4 ligase + 9µL of T4 ligase buffer.
Add 2µL / tube.
Incubation 1h at RT.
Electroporation
Electroporation cuvettes = 2mm ; inoculums of electrocompetent E.coli DH5alpha= 40µL; pulse = 2,5KVolt ; 1h of incubation.
Spread 1mL of inoculums into a petri dish with LB + ampicillin (50mg/mL) (20/0,02mL).